Protein containing tobacco leaf extracts

ABSTRACT

The invention relates to a tobacco leaf extract containing, relative to the total weight of the extract, at least 5 wt.-% proteins essentially free of molecules with a molecular mass of less than 10 kDa. The invention also relates to a pharmaceutical composition containing such an extract and to the use thereof in the treatment of tobacco addiction.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a U.S. National Stage Application pursuant to 35U.S.C. § 371 of International Patent Application PCT/EP2017/058398,filed on Apr. 7, 2017, and published as WO 2017/174787 on Oct. 12, 2017,which claims priority to French Patent Application 1653079, filed onApr. 7, 2016, all of which are incorporated herein by reference in theirentireties for all purposes.

FIELD OF THE INVENTION

The present invention concerns a tobacco leaf extract, as well as apharmaceutical composition containing this tobacco leaf extract and theuse thereof in the treatment of tobacco addiction.

PRIOR ART

In general, dependence or addiction has been defined by the World HealthOrganization as “a syndrome for which the use of a substance takes on amuch higher priority than other behaviours that once had greater value.In its extreme form, the state of dependence is characterized by anirresistible need for a substance that compels the individual sufferingfrom this dependence to impulsively seek this substance”.

Tobacco dependence causes major public health problems, with a largenumber of smoking-related diseases such as cancer (lung, throat, mouth,lips, etc.), cardiovascular diseases, chronic bronchitis, etc. Inaddition to the diseases themselves, smoking has many side effects(decreased fertility, alteration of the epidermis, alteration of theoral and nasal mucosa, alteration of the cerebral arteries, damage tothe oesophagus and stomach, vitamin B and C deficiencies, etc.).

There are three types of tobacco dependence which act concomitantly:physical dependence which is essentially due to the presence of nicotinein tobacco and which is expressed as a feeling of craving (strong urgeto smoke, irritability, nervousness, restlessness, anxiety, sleepdisturbance, etc.); psychological dependence which is inked to thepsychoactive effects of nicotine which provides pleasure, relaxation,intellectual stimulation, anxiolytic, antidepressant and appetitesuppressant action; and environmental or behavioural dependence whichdepends on social and friendly pressure. Physical dependence disappearson average in a few weeks. Withdrawal symptoms peak around three to fourdays after quitting smoking and may last several weeks. Psychologicaldependence takes longer to diminish and can last several months.

Tobacco dependence is maintained through positive and negativereinforcements. Nicotine is responsible for the release of dopamine, aneurotransmitter in the reward circuit. The smoker smokes both toreproduce the feelings of pleasure caused by the release of dopamine(this is the positive reinforcement of the cigarette) and to avoidwithdrawal symptoms when his dopamine level is too low (this is thenegative reinforcement of the cigarette).

There are three main methods of pharmacological treatment of tobaccodependence. The first aims at immediate cessation as of a target quitdate initially set, with pharmacological treatments administered afterthis target quit date in the case of nicotine substitutes and one weekbefore in the case of bupropion or varenicline (in order to reach asufficient plasma concentration at the time of the target quit date).The second consists in reducing his consumption before an attempt tostop, in which the reduction phase corresponds to a pre-treatment duringwhich pharmacological treatments are administered. The third is relapseprevention after successful cessation. To these three methods can beadded cutting down without attempting to stop.

The efficacy of each of these methods is reinforced by a preparationphase for the smoker, who must convince himself of the need to stopsmoking and to reinforce his motivation to do so. The prescribinginformation for pharmacological treatments for tobacco dependence thusindicate that smoking cessation treatments are more likely to besuccessful in patients who are motivated to quit smoking.

There are currently four main types of treatment for tobacco dependence(not to mention behavioural psychotherapies, acupuncture and hypnosis),namely nicotine substitutes, bupropion, varenicline and homeopathy. Theprinciple of action of each of these four methods is based on the factthat nicotine is the molecule responsible for the mechanism of tobaccodependence.

Nicotine substitutes can be administered in a variety of ways:transdermally in the form of patches, orally in the form of chewing gum,lozenges or sublingual tablets, or via the pulmonary route in the formof inhalers. The administration of nicotine substitutes chiefly allowsthe reduction of the negative reinforcements of the cigarette bypreventing as much as possible the smoker from feeling the withdrawalsymptoms related to cessation, with the nicotine provided by thesubstitutes compensating for that of the cigarettes. The efficacy ofnicotine substitutes varies according to the method used, with cessationrates generally ranging from 15 to 20%. Nicotine substitutes are usedduring attempts at immediate cessation and also as pre-treatment beforecessation attempts to help smokers reduce their cigarette consumption aspart of a gradual cessation (cut down to quit). They can also be usedfor the sole purpose of reducing tobacco consumption. Their efficacy inpreventing relapse has not yet been demonstrated.

Bupropion, marketed by GLaxoSmithKine Laboratories (under the brand nameZyban®), acts on certain brain neurotransmitters such as catecholamines,noradrenaline and dopamine. Bupropion is a selective catecholaminereuptake inhibitor, which gives it antidepressant properties. Itsefficacy is equivalent to that obtained after the use of nicotinepatches (cessation rate around 20%). It is prescribed less than nicotinesubstitutes because it has a lower benefit/risk ratio for comparableefficacy. Indeed, it can lead to suicide attempts.

Varenicine, marketed by Pfizer Laboratories (under the brand nameChantix® or Champix®), is a partial agonist of nicotinic acetylcholinereceptors. Varenicline targets these receptors with a dual action: anα4β2 receptor partial agonist, it produces a reaction similar to thatinduced by nicotine with a lower intensity (and thus reduces cravingeffects during withdrawal); and at the beginning of treatment, when thesmoker is treated while smoking occasionally, it attenuatesneurochemical stimulation in the presence of nicotine (α4β2 receptorpartial antagonist). Varenicline thus reduces the negative (withdrawaleffect) and positive (urge to smoke) reinforcements of cigarettes. Inpractice, varenicline reduces the pleasure of smoking, which would be acriterion for future success in quitting smoking. Its efficacy isgreater than that of nicotine substitutes (cessation rate of about 28%).Varenicline is used for immediate cessation attempts and also aspre-treatment for a few weeks prior to cessation attempts to helpsmokers reduce their cigarette consumption as part of a gradualcessation (cut down to quit). In a first study (NCT00789074), 35% ofsmokers were able to reduce their cigarette consumption by at east 50%according to a point made after 3 weeks of pre-treatment withvarenicline. In a second study (NCT00835900), the percentage reductionin the number of cigarettes smoked according to a point made at 4 weekspre-treatment with varenicline was 42%. In a third study (NCT01370356),47% of smokers were able to reduce their cigarette consumption by ateast 50% according to a point made after 4 weeks of pre-treatment withvarenicline. Its efficacy in preventing relapse has not yet beendemonstrated. However, varenicline induces frequent side effects(nausea) and may be the cause of heart problems or suicide attempts.These side effects mean that despite an efficacy superior to nicotinesubstitutes, varenicline is often prescribed only as second-line therapyafter failure with nicotine substitutes.

The fourth cessation route is homeopathy, which is based on the use ofinfinitesimal doses of the substance causing the symptoms one wishes tocontrol. An extract of “tabacum” is often used in smoking cessation.Irish patent application IE 960 511 notably describes the use ofhomeopathic dilutions of tobacco extract for the manufacture of amedicinal product intended for the restoration of neuronal functions andthe relief of nicotine withdrawal symptoms. Like other unconventionalcessation techniques, its efficacy is inadequate in heavy smokers.

Despite much research in this area, there is still a need to find newtreatments or to improve existing treatments for tobacco dependence.

In application PCT/FR2007/000786, the Applicant discovered that giving asmoker an injection of an aqueous solution of extract of tobacco eavesmade it possible to treat the tobacco dependence of said smoker. Thisdiscovery was truly unexpected because the Applicant was able todetermine that said aqueous tobacco leaf extract contained small amountsof nicotine. Thus, the mechanism of action involved in the treatment oftobacco dependence using this aqueous tobacco leaf extract is not thesupply of nicotine to the smoker.

However, the fact that said aqueous tobacco leaf extract contained smallamounts of nicotine did not provide information about its compositionand its method of manufacture. The nicotine content of aqueous tobaccoleaf extracts can indeed vary considerably depending on many factors.

The nicotine content in tobacco eaves can vary from 0.5 to 8% for theprincipal types of tobacco grown (Davis et al., “Production, Chemistry,and Technology”, ISBN-13: 978-0632047918, Chapter 8, page 275).

Burley tobacco contains about twice as much nicotine as Maryland tobaccoand three times as much nicotine as Oriental tobacco (Leffingwell,“Chemical constituents of tobacco leaf and differences among tobaccotypes”, Leffingwell Reports, Vol. 1 (No. 2), February, 2001).

The use of nitrogen fertilizers influences the nicotine content oftobacco eaves and the nicotine content is higher in the upper eaves thanin the middle eaves, which in turn is higher than in the lower eaves(Xiao-Tang et al., “Yield and nicotine content of flue-cured tobacco asaffected by soil nitrogen mineralization”, Pedosphere 18(2): 227-235,2008, ISSN 1002-0160/CN 32-1315/P).

Crop rotation influences the nicotine content of tobacco eaves (Butoracet al., “The Effect of Tobacco Monoculture and Crop Rotations on TobaccoLeaf Composition”, Agriculturae Conspectus Scientificus, Vol. 69 (2004)No. 4 (95-101)).

The chemical composition and nicotine content of tobacco eaves areinfluenced by ultraviolet radiation and light intensity (Andersen etal., “Chemical composition of tobacco eaves altered by near-ultravioletand intensity of visible light”, Plant Physiol. (1973) 51, 723-726).

Extract preparation methods also influence nicotine content,particularly maceration times and conditions, sterilization methods, andphysical and chemical separations.

A tobacco extract such as that described in applicationPCT/FR2007/000786 comprises molecules of low molecular mass, i.e. lessthan 10 kDa. These low molecular weight molecules includetobacco-specific N-nitrosamines (TSNA) and N-nitrosoamino acids derivedfrom non-volatile alkaloids, volatile aldehydes, polynuclear aromatichydrocarbons (such as benzo[a]pyrene), Lactones, urethanes, or metalssuch as cadmium for example (see Wagner et al., Plant Physiol. 1986, 82,274-279 and IARC Monographs on the Evaluation of Carcinogenic Risks toHuman, vol. 49). These compounds can be carcinogenic, mutagenic or toxicand can cause cancer as well as kidney and bone problems. Thesecompounds are thus harmful to the health of the patient to which acomposition containing said compounds has been administered.

Such a tobacco extract comprises many proteins of all molecular weights.For example, the Swiss-Prot database identifies 759 tobacco proteins.The RuBisCO protein alone can represent 30 to 50% of soluble proteins. Acomplete RuBisCO protein typically has eight subunits forming a proteincomplex of about 540 kDa. The toxicity of these proteins administeredsubcutaneously in solution is unknown although there is no indicationthat all or some of them may be involved in any therapeutic effect.

The objective of the present invention is to provide effective treatmentfor tobacco addiction. Advantageously, this treatment is non-toxic, iswell tolerated by patients, and presents a limited risk by reducing asmuch as possible the administration of molecules of unknown toxicitythat do not in principle have a therapeutic effect.

Although tobacco extracts contain thousands of compounds, the Applicantsurprisingly discovered that proteins from tobacco leaf extracts inducea specific IgG immune response that promotes smoking cessation. Thus,the tobacco leaf extract according to the invention comprising a highprotein content and a particular protein composition induces a specificIgG immune response that promotes smoking cessation.

This discovery is all the more surprising as the aqueous tobacco leafextracts contain thousands of compounds, with proteins constituting onlyone family of compounds among others. Tobacco proteins are not known toplay a role in tobacco addiction and therefore there is no incentive touse them in smoking cessation.

The Applicant shows in particular that the tobacco leaf extractsaccording to the invention have a high protein content and a particularprotein composition. This particular protein composition can be notablyobtained when cured tobacco eaves are used. Curing promotes thedegradation of proteins, for example through hydrolysis mechanisms, andthe increase of free amino acids in tobacco eaves (Hamilton &t Lowe,1978; Long &t Weybrew, 1981; Burton, et al., 1983).

The Applicant surprisingly discovered that proteins from tobacco leafextracts according to the invention induce the generation of specificIgG antibodies that promote smoking cessation. The Applicant shows thatthe administration of the tobacco leaf extract according to theinvention induces the specific IgG immune response and makes it possibleto obtain effective treatment for tobacco addiction, even without addingto the extract adjuvant that can promote an immune response. TheApplicant shows that the specific IgG immune response, the treatment oftobacco addiction, and smoking cessation are particularly effective whenextracts of cured tobacco eaves are used.

This discovery is all the more surprising as there is nothing in theprior art to indicate that IgG antibodies specific to tobacco leafextract proteins are likely to help smokers reduce or stop smoking. Theactivation of a specific IgG immune response following theadministration of tobacco extracts and the role of tobacco extracts insmoking cessation have never been described to date. Such a ink betweenthe immune system (involved notably in the production of specific IgGantibodies) and the nervous system (involved notably in the perceptionof the act of smoking and behaviour) which can promote smoking cessationhas never been described to date.

Advantageously, the treatment of tobacco addiction according to thepresent invention requires the administration of the pharmaceuticalcomposition according to the invention only a few times, advantageouslyonly one administration, so that the effects of the reinforcements ofthe cigarette are no longer felt or are felt significantly less by thepatient, and in particular the urge to smoke (positive reinforcement)and the palatability of the cigarette is reduced.

SUMMARY OF THE INVENTION

In a first aspect, the present invention concerns a tobacco leaf extractcontaining at east 5% by weight, based on the total weight of the dryextract, of proteins of molecular mass greater than 10 kDa andessentially free of molecules of molecular mass less than 10 kDa.Preferably, said proteins are selected from the group consisting of thefollowing protein families: lignin-forming anionic peroxidase, glucanendo-1,3-beta-glucosidase, endochitinase, pathogenesis-related protein,osmotin and proteinase inhibitor and mixtures thereof.

The invention further concerns a pharmaceutical composition comprisingsaid tobacco leaf extract and the use thereof in the treatment oftobacco addiction.

The invention further concerns a pharmaceutical composition comprisingsaid tobacco leaf extract and the use thereof in smoking cessation.

The invention further concerns a method for treating tobacco addictioncomprising administering said tobacco leaf extract to a subjectsuffering from tobacco addiction or dependence.

The invention further concerns a method for treating tobacco addictioncomprising administering said pharmaceutical composition comprising saidtobacco leaf extract to a subject suffering from tobacco addiction.

In the context of the present invention, the treatment of tobaccodependence or addiction encompasses all forms of tobacco consumption,namely smoked tobacco, in the form of manufactured or rolled cigarettes,cigarillos or cigars, pipe tobacco, but also smokeless tobacco such assnuff, chewing tobacco or snus.

The invention also concerns the process for preparing said tobacco leafextract containing at east 5% by weight, based on the total weight ofthe dry extract, of proteins of molecular mass greater than 10 kDa andessentially free of molecules of molecular mass less than 10 kDa,comprising the following steps:

-   -   a. Curing of tobacco eaves,    -   b. Grinding of cured tobacco eaves to obtain ground cured        tobacco eaves,    -   c. Extraction of the ground cured tobacco eaves under mechanical        stirring with a solvent, for example an aqueous solvent,        preferably an aqueous buffer solution with a pH between 6.0 and        8.5,    -   d. Separation of solid residues from the ground cured tobacco        leaf extract solution by filtration or centrifugation to obtain        a solid residue-free ground cured tobacco leaf extract solution,    -   e. Constant-volume filtration of the solution obtained in step d        with a solvent, preferably an aqueous solvent, in an amount        ranging from 2 to 12 times by volume, preferably from 3 to 10        times by volume, preferably from 4 to 8 times by volume,        preferably 6 times by volume, based on the volume of the        extract, and with a 10 kDa cut-off membrane,    -   f. Optionally Lyophilization of the protein solution obtained in        step e.

The invention also concerns a kit comprising a pharmaceuticalcomposition comprising said tobacco leaf extract containing at east 5%by weight, based on the total weight of the dry extract, of proteins ofmolecular mass greater than 10 kDa and essentially free of molecules ofmolecular mass less than 10 kDa.

DESCRIPTION OF THE FIGURES

FIG. 1. Illustration of an electrophoretic profile of the tobacco leafextract according to example 1: Fractionation on a 4%-12% NuPage gel inMES buffer and in the presence of reducing agent.

FIG. 2. IgG induction after injection of the tobacco leaf extractaccording to example 1 in mice.

FIGS. 3A-3I. Illustration of immunophenotyping for the characterizationof activated immune cells.

FIG. 4. Activation of natural killer cells by the tobacco leaf extractaccording to example 1.

FIG. 5. Activity of the tobacco leaf extract according to example 1 on Tand B lymphocytes.

FIG. 6. Induction of pro-inflammatory cytokines and of IFNγ by thetobacco leaf extract according to example 1.

FIG. 7. Induction of TH2 cytokines and of chemokines and hematopoieticgrowth factor by the tobacco leaf extract according to example 1.

FIGS. 8 and 9. Change in tobacco consumption in patients treated byadministration of the tobacco leaf extract according to example 1.

DETAILED DESCRIPTION OF THE INVENTION

Tobacco Leaf Extract

The present invention concerns a tobacco leaf extract containing at east5% by weight, based on the total weight of the dry extract, preferablyat east 10% by weight, preferably at east 15% by weight, preferablyabout 20% by weight of proteins of molecular mass greater than 10 kDaand essentially free of molecules of molecular mass less than 10 kDa.

The dry extract is advantageously obtained before lyophilization of thetobacco leaf extract according to the invention, then placed under avacuum bell-jar, preferably in the presence of P₂O₅, until a constantmass of extract is obtained.

Advantageously, the content of molecules of molecular mass less than 10kDa is less than 5% by weight based on the total weight of the extract,preferably less than 2.5% by weight, and better still less than 1% byweight.

For the purposes of the present invention, “about” means plus or minus1% due to measurement uncertainties.

The content of proteins of molecular mass greater than 10 kDa usuallymeasured in tobacco leaf extracts is about 1%. The tobacco leaf extractaccording to the present invention therefore has a content of proteinsof molecular mass greater than 10 kDa which is much higher thanpreviously known.

Preferably, the proteins present in the extract according to the presentinvention are chosen from the group consisting of the following proteinfamilies: lignin-forming anionic peroxidase, glucanendo-1,3-beta-glucosidase, endochitinase, pathogenesis-related protein,osmotin and proteinase inhibitor and mixtures thereof.

The names indicated for the proteins present in the extract according tothe present invention correspond to the names given in the Swiss-Protdatabase, which is a biological database listing protein sequences.

According to one embodiment, the tobacco leaf extract according to theinvention comprises at east one protein belonging to the family ofglucan endo-1,3-beta-glucosidases, and preferably chosen frombeta-1,3-endoglucanase acidic isoform PR-Q′ (PR36401 according to theUniProt database), beta-1,3-endoglucanase basic vacuolar isoform GLB(P27666 according to the UniProt database), and mixtures thereof.

According to one embodiment, the tobacco leaf extract according to theinvention comprises at east one protein belonging to the family ofendochitinases, and preferably chosen from acidic endochitinase P(P17513 according to the UniProt database), acidic endochitinase Q(P17514 according to the UniProt database), endochitinase B (P24091according to the UniProt database), and mixtures thereof.

According to one embodiment, the tobacco leaf extract according to theinvention comprises at east osmotin (P14170 according to the UniProtdatabase).

According to one embodiment, the tobacco leaf extract according to theinvention comprises at east one lignin-forming anionic peroxidase(P11965 according to the UniProt database).

According to one embodiment, the tobacco leaf extract according to theinvention comprises at east one pathogenesis-related protein, andpreferably chosen from pathogenesis-related protein R (P13046 accordingto the UniProt database), pathogenesis-related protein PR-4A (PR29062according to the UniProt database), pathogenesis-related protein PR-4B(PR29063 according to the UniProt database), and mixtures thereof.

According to one embodiment, the tobacco leaf extract according to thepresent invention comprises at east one protein belonging to the familyof proteinase inhibitors, and preferably chosen from proteinaseinhibitor I-B (Q03199 according to the UniProt database), proteinaseinhibitor I-A (Q03198 according to the UniProt database), and mixturesthereof.

According to one embodiment, the tobacco leaf extract also comprisespolysaccharides of molecular mass greater than 10 kDa and preferablywater-soluble.

Preferably, the tobacco leaf extract according to the present inventioncomprises at east 5% by weight based on the total weight of the dryextract, preferably at east 10% by weight, preferably at east 15% byweight, preferably about 20% by weight of proteins selected from thegroup consisting of the following protein families: lignin-forminganionic peroxidase, glucan endo-1,3-beta-glucosidase, endochitinase,pathogenesis-related protein, osmotin and proteinase inhibitor andmixtures thereof.

According to one embodiment, the tobacco leaf extract according to thepresent invention is essentially free of high molecular mass proteins.

For the purposes of the present invention, “high molecular mass protein”means a protein whose molecular mass is greater than 500 kDa, preferablywhose molecular mass is greater than 400 kDa, more preferably whosemolecular mass is greater than 300 kDa, preferentially whose molecularmass is greater than 200 kDa, even more preferentially whose molecularmass is greater than 150 kDa, and better still whose molecular mass isgreater than 100 kDa.

According to one embodiment, the tobacco leaf extract according to thepresent invention is essentially free of proteins whose molecular massis greater than 50 kDa.

For the purposes of the present invention, “substantially free” means acontent of molecules less than 15% by weight based on the total proteinweight of the extract, preferably a content of molecules less than 10%by weight based on the total protein weight of the extract, morepreferably less than 7.5% by weight based on the total protein weight ofthe extract, more preferably less than 5% by weight based on the totalprotein weight of the extract, preferentially less than 2.5% by weightbased on the total protein weight of the extract, more preferentiallyless than 1% by weight based on the total protein weight of the extract,and better still less than 0.5% by weight based on the total proteinweight of the extract.

Advantageously, the content of high molecular mass proteins is less than15% by weight based on the total protein weight of the extract,preferably less than 10% by weight based on the total protein weight ofthe extract, more preferably less than 7.5% by weight based on the totalprotein weight of the extract, more preferably less than 5% by weightbased on the total protein weight of the extract, preferentially lessthan 2.5% by weight based on the total protein weight of the extract,more preferentially less than 1% by weight based on the total proteinweight of the extract, and better still less than 0.5% by weight basedon the total protein weight of the extract.

According to one embodiment, the tobacco leaf extract according to thepresent invention is essentially free of RuBisCO proteins.

According to one embodiment, the tobacco eaves are of the varietyNicotiana Tabacum or the variety Nicotiana Rustica. The tobacco eavesmay come from brown or blond tobacco and may be selected from Virginiatobacco, Burley tobacco, Oriental tobacco, Latakia tobacco, Periquetobacco, Maryland tobacco, Kentucky tobacco, California tobacco, Tex Mextobacco, and mixtures thereof.

Of course, the extract does not necessarily consist of a pure extract oftobacco eaves, and proteins extracted from cannabis can be added totreat both tobacco and cannabis dependence.

According to a particular embodiment, the tobacco leaf extract isobtained from a 1/1/1 blend of brown tobacco, Virginia tobacco andBurley tobacco.

According to a particular embodiment, the tobacco leaf extract isobtained from Burley tobacco.

According to one embodiment, the tobacco leaf extract is obtained fromcured tobacco eaves.

According to a particular embodiment, the tobacco leaf extract isobtained from air-cured tobacco eaves.

According to a particular embodiment, the tobacco leaf extract isobtained from tobacco eaves air-cured for a duration adapted to localclimatic conditions, preferably for a minimum duration of one month.

For the purposes of the present invention, “air-curing” or “naturalcuring” means curing carried out in the presence of natural outdoor orindoor air. Air-curing can be carried out in an open or closed, coveredor uncovered structure. Air-curing can for example be carried out in anatural curing barn. In this case, freshly harvested or pre-curedtobacco eaves are left to cure naturally under the effect of open air.The tobacco eaves can for example be hung in unheated ventilated barns.The tobacco eaves can for example be left to cure naturally until theyturn brown. At this stage, there is practically no sugar left in theleaf. Advantageously, curing is carried out for a duration adapted tolocal climatic conditions, preferably for a minimum duration of onemonth. By way of example, curing can be carried out between Septemberand December for harvests in central France. The tobacco eaves may beturned or aerated one or more times during curing, so as to allowuniform curing and to avoid the formation of condensation and therotting or degradation of the eaves. The air-curing method can be usedfor example for Burley variety tobacco.

According to a particular embodiment, the extract of tobacco eaves isobtained from tobacco eaves cured in a natural curing barn.

According to another particular embodiment, the tobacco leaf extract isobtained from tobacco eaves cured in a heated curing barn. In this case,curing can be carried out in a barn or a structure heated to a suitabletemperature.

According to another particular embodiment, the tobacco leaf extract isobtained from flue-cured tobacco eaves. In this case, curing can becarried out in a structure or a barn heated to a suitable temperature.Heat can be introduced into the structure or the barn through ductsconnected to an external heat source. This controlled heating producesyellow-orange eaves. These eaves thus contain a high sugar content.Virginia tobacco can for example be cured according this method.

According to another particular embodiment, the tobacco leaf extract isobtained from sun-cured tobacco eaves. In this case, the tobacco eavescan be spread on racks and exposed to the sun for 12 to 30 days. Underdirect light and heat from the sun, the eaves turn yellow or orange andretain a high sugar content. Oriental tobacco is generally cured usingthis method.

According to another particular embodiment, the tobacco leaf extract isobtained from fire-cured tobacco eaves. In this case, small pieces ofwood can be burned beneath the tobacco eaves, which cure while absorbinga “smoky” aroma.

The tobacco leaf extract according to the present invention isadvantageously an aqueous extract.

The tobacco leaf extract according to the present invention isadvantageously an aqueous extract that is brown in colour.

Preferably, the tobacco leaf extract is obtainable by a solventextraction process, for example an aqueous solvent, of ground curedtobacco eaves followed by separation of solid residues from the groundcured tobacco leaf extract solution then constant-volume diafiltrationof the solid residue-free ground cured tobacco leaf extract solutionwith an aqueous solvent in an amount ranging from 2 to 12 times byvolume, preferably 3 to 10 times by volume, preferably 4 to 8 times byvolume, preferably 6 times by volume, based on the volume of the extractand with a 10 kDa cut-off membrane.

Of course, other types of solvent can be used to carry out theextraction process of the tobacco leaf extract. The extraction of step ccan be carried out with an organic or inorganic solvent, or with amixture of organic and/or inorganic solvents.

To ensure good extraction efficiency and yield, and to obtain an extractof good quality with the desired purity properties and composition, theskilled person will easily know how to select the appropriate solvent(s)according to the following criteria:

-   -   the polarity of the solvent or solvent mixture (polar or        non-polar);    -   the physical state of the solvent or solvent mixture (for        example liquid, solid, supercritical or gaseous);    -   the chemical nature of the solvent or solvent mixture (for        example organic or inorganic);    -   the charge of the solvent or solvent mixture (for example ionic        or non-ionic);    -   the origin of the solvent or solvent mixture;    -   the miscibility of the solvent or solvent mixture;    -   the solubility of the solvent or solvent mixture.

The skilled person will know which technique to use to select thesolvent(s) corresponding to his criteria, based on the desiredextraction yield and extract purity and composition.

Advantageously, the tobacco leaf extract according to the presentinvention is obtained by the tobacco leaf preparation process describedbelow.

Pharmaceutical Composition

In a second aspect, the present invention concerns a pharmaceuticalcomposition comprising as active principle the tobacco leaf extract asdescribed above.

The pharmaceutical composition according to the present inventionfurther comprises at east one pharmaceutically acceptable excipient,such as pharmaceutically acceptable solvents, and for example water.

Advantageously, the pharmaceutical composition comprises proteinspresent in the tobacco leaf extract according to the invention in acontent ranging from 1 to 1 000 μg/mL, preferably from 10 to 500 μg/mL,preferably from 50 to 300 μg/mL, preferably from 60 to 200 μg/mL,preferably between 80 and 150 μg/mL.

The pharmaceutical composition according to the invention is preferablyan aqueous composition.

According to a particular embodiment, the pharmaceutical composition mayfurther comprise an active agent acting on negative reinforcement, suchas for example a nicotine substitute, varenicline or bupropion.

According to an embodiment, the pharmaceutical composition furthercomprises an adjuvant. Examples of adjuvants include swelling agentssuch as for example a sugar such as lactose, sucrose, trehalose,sorbitol, glucose, raffinose, mannitol, preferably lactose, sucrose,trehalose, glucose, or mannitol, an amino acid such as arginine, glycineor histidine, preferably glycine, or polymers of dextran or polyethyleneglycol type, or mixtures thereof. According to this embodiment, thepharmaceutical composition comprises 50 to 99% by weight of adjuvants,preferably 80 to 97% by weight, based on the total weight of thepharmaceutical composition.

According to a particular embodiment, the pharmaceutical composition mayfurther comprise proteins extracted from cannabis.

According to one embodiment, the pharmaceutical composition according tothe present invention is provided in a form suitable for subcutaneousadministration.

According to another embodiment, the pharmaceutical composition isprovided in a form suitable for administration by means of an adhesivetransdermal therapeutic system. Advantageously, the pharmaceuticalcomposition is provided in patch form.

According to another embodiment, the pharmaceutical composition isprovided in a form suitable for administration by spraying or byvaporization. Advantageously, the pharmaceutical composition is providedin sprayer, vaporizer or aerosol form.

The pharmaceutical composition according to the invention is preferablyprepared according to the process for preparing a pharmaceuticalcomposition comprising the tobacco leaf extract described below.

Use of the Pharmaceutical Composition

In a third aspect, the present invention concerns the use of thepharmaceutical composition for the treatment of tobacco addiction.

For the purposes of the present invention, “treatment of tobaccoaddiction” or “treatment of tobacco dependence” means more precisely thereduction of the addictive reinforcements of the cigarette. Theadministration of the pharmaceutical composition according to theinvention notably reduces the positive reinforcements of the cigaretteand in particular the urge to smoke.

The present invention also concerns a composition comprising a tobaccoleaf extract as defined above for use in the treatment of tobaccoaddiction.

In other words, the present invention also concerns a method fortreating tobacco addiction or dependence, comprising administering acomposition comprising a tobacco leaf extract as defined above.

Advantageously, the administration of the composition according to theinvention enables the patient to stop smoking, to decrease hisconsumption or to prevent his relapse after stopping by attenuating thepositive reinforcements of tobacco and notably the urge to smoke, thusfacilitating smoking cessation. Advantageously, the administration ofthe composition helps any type of smoker, i.e. including heavy smokers,to stop smoking and to decrease or avoid relapse after quitting.

Advantageously, the administration of the composition according to theinvention further comprising proteins extracted from cannabis makes itpossible to treat dependence or addiction to tobacco and cannabis at thesame time.

According to one embodiment, the composition according to the presentinvention is administered by subcutaneous injection. According to thisembodiment, the pharmaceutical composition is provided in a formsuitable for subcutaneous administration.

According to one embodiment, the pharmaceutical composition is providedin a dosage form of 0.03 mL to 10 mL, preferably 0.1 mL to 5 mL,preferably 0.5 to 2 mL. The pharmaceutical composition is thuspreferably administered in an amount of 0.03 mL to 10 mL, preferably 0.1to 5 mL, preferably 0.5 mL to 2 mL by subcutaneous injection.

According to one embodiment, the composition according to the presentinvention is administered by means of an adhesive transdermaltherapeutic system containing the tobacco leaf extract as defined above.According to this embodiment, the pharmaceutical composition is providedin a form suitable for administration by means of an adhesivetransdermal therapeutic system. Advantageously, the pharmaceuticalcomposition is provided in patch form.

The patch can be in the form of a reservoir-type patch with one or morecompartments or a matrix-type patch. The practical execution of thepatches will be determined by the skilled person based on his generalknowledge of the subject to obtain a controlled and prolonged systemicadministration of the tobacco leaf extract, over the entire period ofapplication of the patch, for example for a duration of about 2h to 24h.

The reservoir-type patch will comprise one or more separate reservoir(s)containing the active principle(s) (including the tobacco leaf extract)in solution or suspended in the polymer matrix coming into contact withthe skin via a semi-permeable polymeric membrane allowing the rate ofrelease of the active principle(s) to be adjusted.

The matrix-type patch will comprise a polymeric mass within which theactive principle(s) will be dissolved or dispersed in the appropriateproportions. These active principles are released by diffusion throughthe polymer chains of said matrix.

According to a particular embodiment of this type of patch, the adhesivecovers the entire release surface of the matrix and is an integral partof the latter. This is therefore an active adhesive-type patch wellknown to the skilled person, which is of simplified manufacture andwhich allows the creation of thin, suitably flexible patches allowingcomfortable application on the patient's skin.

To ensure a good infusion of these active principles subcutaneously, orin the bloodstream, at the appropriate dose, at the appropriatediffusion rate, and for the appropriate duration, the skilled person caneasily set the following parameters:

-   -   the ratio of the surface areas and the volumes of each        compartment of the patch;    -   the optional addition of one or more hydrophilic additive(s);    -   the optional addition of one or more diffusion activator(s) or        inhibitor(s);    -   the optional addition of one or more solubilizer(s);    -   the optional addition of one or more stabilizer(s);    -   the optional addition of one or more absorption promoter(s),        and;    -   more generally, all types of additives well known to the skilled        person allowing good control of the flow and stability of the        tobacco leaf extract.

The skilled person will know which technique to use to set theparameters listed above based on the desired solubility and stability.

The various manufacturing parameters of the patch will be easily adaptedby the skilled person to arrive at the desired dosage.

In a manner known per se, such a patch comprises a removable protectivefilm which is intended to preserve the adhesive side to be applied tothe skin after the patch has been manufactured and throughout itsstorage period. In a manner known per se, the skilled person will forexample use polyester films, one side of which can be treated withanti-adherent silicones.

According to another embodiment, the composition according to thepresent invention is administered by means of a spraying or vaporizationsystem containing the tobacco leaf extract as defined above. Accordingto this embodiment, the pharmaceutical composition is provided in a formsuitable for administration by spraying or vaporization. Advantageously,the pharmaceutical composition is provided in sprayer, vaporizer oraerosol form.

The sprayer, vaporizer or aerosol can be in the form of a sprayer,vaporizer or aerosol having a reservoir with one or more compartments.The practical execution of the sprayers, vaporizers or aerosols will bedetermined by the skilled person based on his general knowledge of thesubject to obtain a controlled and prolonged systemic administration ofthe tobacco leaf extract.

The sprayer, vaporizer or aerosol will have one or more separatereservoir(s) containing the active principle(s) (including the tobaccoleaf extract) in solution or suspension. The active principle(s) is/arethen administered by spraying or vaporization on the area of the body tobe treated. Spraying or vaporization can for example be carried out onthe skin. The active principle(s) may be administered by spraying orvaporization into the mucous membranes.

To ensure a good infusion of these active principles subcutaneously, orin the bloodstream, at the appropriate dose, at the appropriatediffusion rate, and for the appropriate duration, the skilled person caneasily set the following parameters:

-   -   the ratio of the surface areas and the volumes of each        compartment or reservoir of the sprayer, vaporizer or aerosol;    -   the optional addition of one or more hydrophilic additive(s);    -   the optional addition of one or more diffusion activator(s) or        inhibitor(s);    -   the optional addition of one or more solubilizer(s);    -   the optional addition of one or more stabilizer(s);    -   the optional addition of one or more absorption promoter(s),        and;    -   more generally, all types of additives well known to the skilled        person allowing good control of the flow and stability of the        tobacco leaf extract.

The skilled person will know which technique to use to set theparameters listed above based on the desired solubility and stability.

The different manufacturing parameters of the sprayer, vaporizer oraerosol will be easily adapted by the skilled person to arrive at thedesired dosage.

According to a first embodiment, the pharmaceutical composition isprovided in a form intended to be administered only once. According tothis embodiment, the pharmaceutical composition is administered onlyonce, thus allowing a reduction or a suppression of the positivereinforcements of tobacco and notably the urge to smoke.

According to another embodiment, the pharmaceutical composition isprovided in a form intended to be administered several times, preferablytwo or three times. According to this embodiment, the pharmaceuticalcomposition is preferably administered on days 0 and 10 or on days 0, 10and 30, thus allowing a reduction or a suppression of the positivereinforcements of tobacco and notably the urge to smoke. In thisembodiment, the dose injected the second time, and optionally the thirdtime, is identical to the dose injected the first time.

According to a particular embodiment, the pharmaceutical compositionaccording to the present invention may be administered with acomposition comprising an active agent acting on negative reinforcement,such as a nicotine substitute, varenicline, bupropion or mixturesthereof. According to this embodiment, several specific modes ofadministration can be envisaged:

-   -   (i) administration, prior to the attempt to stop smoking, of the        pharmaceutical composition according to the invention in order        to reduce cigarette consumption, followed by administration of        the active agent acting on negative reinforcement in order to        limit the effects of the negative reinforcement, and in        particular the sensation of withdrawal;    -   (ii) concomitant administration of the pharmaceutical        composition according to the invention and of an active agent        acting on negative reinforcement at the time of the attempt to        stop smoking, with the aim of immediate cessation;    -   (iii) administration at the time of the attempt to stop smoking        of an active agent acting on negative reinforcement, for example        for 12 weeks after the attempt to stop, followed by        administration of the pharmaceutical composition according to        the invention, for example for 2 weeks after the end of the        administration of the active agent acting on negative        reinforcement, with the aim of preventing relapses.

Preparation of the Tobacco Leaf Extract

In a fourth aspect, the present invention concerns the preparation of atobacco leaf extract according to the present invention.

Advantageously, the process for preparing the tobacco leaf extractaccording to the present invention comprises the following steps:

-   -   a. Curing of tobacco eaves,    -   b. Grinding of cured tobacco eaves to obtain ground cured        tobacco eaves,    -   c. Extraction of the ground cured tobacco eaves under mechanical        stirring with a solvent, for example an aqueous solvent,        preferably an aqueous buffer solution with a pH between 6.0 and        8.5,    -   d. Separation of solid residues from the ground cured tobacco        leaf extract solution by filtration or centrifugation to obtain        a solid residue-free ground cured tobacco leaf extract solution,    -   e. Constant-volume filtration of the solution obtained in step d        with a solvent, preferably an aqueous solvent, in an amount        ranging from 2 to 12 times by volume, preferably 3 to 10 times        by volume, preferably 4 to 8 times by volume, preferably 6 times        by volume, based on the volume of the extract, and with a 10 kDa        cut-off membrane,    -   f. Optionally Lyophilization of the protein solution obtained in        step e.

According to a particular embodiment, the curing of step a is carriedout in open air, referred to as natural curing. In this case, curing iscarried out in the presence of natural open air, outdoors or indoors.Curing can be carried out in an open or closed, covered or uncoveredstructure. Air-curing can for example be carried out in a natural curingbarn. In this case, freshly harvested or pre-cured tobacco eaves areleft to cure naturally under the effect of open air. The tobacco eavescan for example be hung in unheated ventilated barns. The tobacco eavescan for example be left to cure naturally until they turn brown. At thisstage, there is practically no sugar left in the leaf. Advantageously,curing is carried out for a duration adapted to local climaticconditions, preferably for a minimum duration of one month. By way ofexample, curing can be carried out between September and December forharvests in central France. The tobacco eaves may be turned one or moretimes during curing, so as to allow uniform curing and to avoid theformation of condensation and the rotting or degradation of the eaves.The air-curing method can be used for example for Burley varietytobacco. According to a particular embodiment, the curing of step a iscarried out in a natural curing barn.

According to another particular embodiment, the curing of step a iscarried out in a heated curing barn. In this case, curing can be carriedout in a barn or a structure heated to a suitable temperature.

According to another particular embodiment, the curing of step a iscarried out by flue curing. In this case, curing can be carried out in astructure or a barn heated to a suitable temperature. Heat can beintroduced into the structure or the barn through ducts connected to anexternal heat source. This controlled heating produces yellow-orangeeaves. These eaves thus contain a high sugar content. Virginia tobaccocan for example be cured using this method.

According to another particular embodiment, the curing of step a iscarried out by sun curing. In this case, the tobacco eaves can be spreadon racks and exposed to the sun for 12 to 30 days. Under direct lightand heat from the sun, the eaves turn yellow or orange and retain a highsugar content. Oriental tobacco is generally cured using this method.According to another particular embodiment, the curing of step a iscarried out by fire curing. In this case, small pieces of wood can beburned beneath the tobacco eaves, which cure while absorbing a “smoky”aroma.

Advantageously, the curing of step a promotes the degradation of highmolecular mass tobacco proteins, such as RuBisCO, for example byhydrolysis mechanisms.

Steps c to e correspond to the extraction, filtration and diafiltrationsteps of the type described in U.S. Pat. No. 5,770,698 (column 6, line47 to column 7, line 7) and patent application US 2009/0162403(paragraphs [0032] to [0040]).

Preferably, the extraction in step c is carried out at a temperaturebetween 4 and 20° C., preferably between 4 and 10° C.

Preferably, the extraction in step c is carried out for 12 to 36 h,preferably 22 to 26h, preferably 24h.

According to a particular embodiment, the solvent used in step c is anaqueous solvent. Preferably, the aqueous solvent used in step c is anaqueous buffer solution of ammonium bicarbonate, preferably at aconcentration between 2 and 6 g/L, preferably 4 g/L.

Of course, other types of solvents can be used to carry out theextraction of step c. The extraction of step c can be carried out withan organic or inorganic solvent, or with a mixture of organic and/orinorganic solvents.

To ensure good extraction efficiency and yield, and to obtain an extractof good quality with the desired purity properties and composition, theskilled person will easily know how to select the appropriate solvent(s)according to the following criteria:

-   -   the polarity of the solvent or solvent mixture (polar or        non-polar);    -   the physical state of the solvent or solvent mixture (for        example liquid, solid, supercritical or gaseous);    -   the chemical nature of the solvent or solvent mixture (for        example organic or inorganic);    -   the charge of the solvent or solvent mixture (for example ionic        or non-ionic);    -   the origin of the solvent or solvent mixture;    -   the miscibility of the solvent or solvent mixture;    -   the solubility of the solvent or solvent mixture.

The skilled person will know which technique to use to select thesolvent(s) corresponding to these criteria, based on the desiredextraction yield and extract purity and composition.

Preferably, the extraction in step c is carried out by suspending theground cured tobacco eaves in a buffer solution, preferably at aconcentration of ground cured tobacco eaves in the buffer solution of 30to 70 g/L, preferably 40 to 60 g/L, and more preferentially 50 g/L. Thesuspended solid residues are then removed by filtration, for example byBüchner filtration (step d), in order to obtain ground cured tobaccoeaves free of solid residue.

Preferably, the aqueous solvent used in step d is water for injection(WFI).

Advantageously, step e removes more than 99% of molecules of molecularmass less than 10 kDa and notably free amino acids, proteins andpeptides of molecular mass less than 10 kDa and protein residues ofmolecular mass less than 10 kDa resulting from the degradation of theproteins of step a.

Preferably, the protein solution obtained in step e is subjected to astep e′ of sterilizing filtration, prior to step f.

The tobacco leaf extract according to the invention obtained at the endof the process described above may be stored as obtained at the end ofstep d′ of sterilizing filtration, or lyophilized as obtained at the endof step f.

Preparation of the Pharmaceutical Composition Comprising the TobaccoLeaf Extract

In a fifth aspect, the present invention concerns the preparation of thepharmaceutical composition comprising the tobacco leaf extract accordingto the invention.

The pharmaceutical composition may be prepared by a process comprisingthe following steps:

-   -   a. Preparation of a tobacco leaf extract as defined above,    -   b. Adjustment of the protein concentration so as to obtain a        protein concentration ranging from 100 to 200 μg/mL,    -   c. Addition of the pharmaceutically acceptable excipient(s), and    -   d. Optionally, addition of adjuvant(s) to this extract,        preferably addition of mannitol.

The tobacco leaf extract used in step b can be either that obtaineddirectly after the step of diafiltration on a 10 kDa cut-off membrane,or that obtained after the step of sterilizing diafiltration, or thetobacco leaf extract which is Lyophilized then reconstituted for examplein saline solution or water.

Advantageously, the concentration in step b can be adjusted:

-   -   either by dilution with water in order to lower the protein        concentration,    -   or by diafiltration in order to increase the protein        concentration.

A pharmaceutically acceptable excipient particularly preferred in step cis water.

The adjuvants that can be added in step d may be notably swelling agentssuch as for example a sugar such as lactose, sucrose, trehalose,sorbitol, glucose, raffinose, mannitol, preferably lactose, sucrose,trehalose, glucose, or mannitol, an amino acid such as arginine, glycineor histidine, preferably glycine, or polymers of the dextran orpolyethylene glycol type; or mixtures thereof.

According to a particular embodiment, step d comprises at east theaddition of mannitol.

Kit Comprising the Pharmaceutical Composition Comprising the TobaccoLeaf Extract

In a sixth aspect, the present invention concerns a kit comprising dosesof the tobacco leaf extract or of the pharmaceutical compositionaccording to the invention.

According to a first embodiment, the kit comprises one or more dose(s)of tobacco leaf extract, preferably in Lyophilized form, as well as oneor more dose(s) of saline solution or of WFI to prepare thepharmaceutical composition according to the invention just beforeadministration.

According to another embodiment, the kit comprises one or more dose(s)of pharmaceutical composition ready to be administered to the patient.

The kit may optionally comprise one or more syringe(s) in order toadminister the pharmaceutical composition by subcutaneous injection.

The kit may optionally comprise one or more patch(s) in order toadminister the pharmaceutical composition transdermally.

The following examples aim to illustrate the present invention.

EXAMPLES Example 1: Preparation of a Tobacco Leaf Extract According tothe Invention

Burley tobacco eaves are used to prepare the extract.

100 g of Burley tobacco eaves cured naturally for about three months (incentral France, between September and December) are ground. The groundeaves are suspended for 24h at a temperature of 4 to 10° C. in 1 892 gof WFI to which 8 g of ammonium bicarbonate has been added. Thesuspended solid residues are then removed by Büchner filtration.

A 0.2 μm clarifying filtration of the extract is then carried out. Theextract obtained is brown in colour. The extract is then weighed todetermine the volume of WFI to be used in the constant-volumediafiltration step; in this case the mass of the liquid extract is 1 680g.

An extraction of the proteins of this solid residue-free ground curedtobacco leaf extract is then performed: 10 080 g of WFI is then added tothe solid residue-free ground cured tobacco leaf extract. The 11 760 gof solution thus obtained is diafiltered at constant volume against 6times the volume with a 10 kDa cut-off (MerckMillipore) until the massof the retentate is reduced to 1 680 g.

The retentate obtained after diafiltration is a protein mixture in whichproteins of molecular mass less than 10 kDa are detected with aconcentration 99% lower than their initial concentration. The colour ofthe retentate obtained is between B2 and B3 according to the measurementscale defined in EUROPEAN PHARMACOPOEIA 5.0, 2.2.2, Degree of colorationof liquids.

This diafiltrate can be Lyophilized, after addition of mannitol forexample, using an SMH 150 Lyophilizer, to obtain a Lyophilized tobaccoleaf extract.

Example 2: Determination of the Protein Composition of the Tobacco LeafExtract Prepared According to Example 1

The proteins of the tobacco leaf extract of example 1 are firstseparated on a polyacrylamide gel. To this end, the extract to beanalysed is added to a NuPage® Bis-Tris gel (4-12%) and electrophoresisis performed at 200 V for 35 min, before being brought into contact with20 mL of Instant Blue for 1h then rinsed with water overnight. Theselected polyacrylamide gel bands are cut out (6 bands) and thendestained with 50 mM NH₄CO₃/CH₃CN (50/50) buffer. The disulphide bridgesare then reduced with 10 mM dithiothreitol/50 mM NH₄CO₃ solution for 40min at 55° C. The reduced cysteines are then alkylated with 100 mMiodoacetamide/50 mM NH₄CO₃ solution for 30 min at room temperature andin the dark.

The protein solution thus obtained is digested with an enzyme to obtainprotein or peptide fragments: the digestion is carried out with trypsin(trypsin V5111, Promega) in an amount adapted to the staining of thepolyacrylamide gel bands in 50 mM NH₄CO₃ solution overnight at 37° C.

The peptides of the peptide digestate are then separated by liquidnanochromatography (U3000 NanoLC System, ThermoFischer Scientific) withpreconcentration on PepMap C18 micro-precolumn (5 μm; 100 Å; 300 μm×5mm; ThermoFisher Scientific) then elution on PepMap C18 nanocolumn (3μm; 100 Å; 75 μm×250 mm; ThermoFisher Scientific) in linear gradientmode; buffer A 0.1% HCOOH in H₂O/CH₃CN (95/5), buffer B 0.1% HCOOH inH₂O/CH₃CN (20/80), Gradient 0 to 60% B in 60 min, flow rate 300 μL/min).

The peptides are then analysed by mass spectrometry on an LTQ Velosinstrument (Dual Pressure Linear Ion Trap; ThermoFisher Scientific)equipped with a nanospray source (ThermoFisher Scientific) and coupledto the U3000 NanoLC device. Data are acquired with the Excalibur 2.1software (ThermoFisher Scientific) in positive mode by cycle of an MSscan from m/z 400 to 1600 in “Resolution Enhanced” mode followed by MSMSscans in “Normal Resolution” mode on the 20 most intense MS ions (charge2 and higher) in CID mode under helium with collision energy of 35 eV.Previously fragmented MS ions are dynamically excluded for 30 s with amass tolerance of 50 mmu.

The masses of the peptides and their fragments are compared withexisting data in the databases for purposes of identification. To thisend, the MS and MSMS data are processed with the ProteomeDiscoverer 1.4software according to the MASCOT search algorithm (Version 2.4) and theUniprotKB/Swiss-Prot database (April 2015 release) reduced to the TOBACspecies is queried.

The identification of the proteins is validated according to theconfidence value p of less than 0.05. Only proteins identified with ateast 2 peptides of maximum confidence are retained.

The scores of each protein thus identified allow only a relativeclassification of proteins between them, this classification beingcorrelated to the concentrations of these proteins.

The absolute values of these scores depend on the operating conditions,the test sample and the amounts deposited.

The list of the 12 proteins thus identified and classified in descendingorder of their scores (from the most present to the east present) is asfollows:

-   -   P36401; E13H_TOBAC; Glucan endo-1,3-beta-glucosidase, acidic        isoform PR-Q′ (EC 3.2.1.39) ((1->3)-beta-glucan endohydrolase)        ((1->3)-beta-glucanase) (Beta-1,3-endoglucanase) (PR-35); MM:        36995 Da    -   P17513; CHIP_TOBAC; Acidic endochitinase P (EC 3.2.1.14)        (Pathogenesis-related protein P) (PR-P); MM: 27469 Da    -   P17514; CHIQ_TOBAC; Acidic endochitinase Q (EC 3.2.1.14)        (Pathogenesis-related protein Q) (PR-Q); MM: 27633 Da    -   P27666; E13F_TOBAC; Glucan endo-1,3-beta-glucosidase, basic        vacuolar isoform GLB (EC 3.2.1.39) ((1->3)-beta-glucan        endohydrolase) ((1->3)-beta-gLucanase) (Beta-1,3-endoglucanase,        basic) (Glucanase GLB); MM: 40443 Da    -   P14170; OSMO_TOBAC; Osmotin; MM: 26681 Da    -   P24091; CHI2_TOBAC; Endochitinase B (CHN-B) (EC 3.2.1.14); MM:        34721 Da    -   P11965; PERX_TOBAC; Lignin-forming anionic peroxidase (EC        1.11.1.7) (TOPA); MM: 34674 Da    -   P13046; PRR1_TOBAC; Pathogenesis-related protein R major form        (Thaumatin-Like protein E22); MM: 24667 Da    -   P29062; PR4A_TOBAC; Pathogenesis-related protein PR-4A; MM:        16221 Da    -   Q03199; IPIB_TOBAC; Proteinase inhibitor I-B (PI-IB) (Inhibitor        of microbial serine proteinases major isoform); MM: 11916 Da    -   Q03198; IPIA_TOBAC; Proteinase inhibitor I-A (PI-IA) (Inhibitor        of microbial serine proteinases minor isoform); MM: 11880 Da    -   P29063; PR4B_TOBAC; Pathogenesis-related protein PR-4B; MM:        16235 Da

The tobacco leaf extract preparation process thus makes it possible toconcentrate certain proteins and to remove others. Thus, of the 759Nicotiana tabacum proteins listed in Swiss-Prot, only 6 families and 12proteins are predominantly present in the tobacco leaf extract preparedaccording to example 1. The 12 proteins that are predominantly presentin the tobacco leaf extract prepared according to example 1 have amolecular mass between 10 and 50 kDa.

Example 3: Determination of Protein Content

The protein content in the tobacco leaf extract according to example 1(before and after the diafiltration step) was determined according tothe Bradford method.

The extract of example 1 is dissolved in saline solution.

Three standard protein solutions were prepared: 50 μL of a 2 mg/mLbovine serum albumin solution (Sigma Aldrich, product number P0834) isdiluted 20 times with 950 μL of saline solution in order to obtainstandardized solutions at exactly 100 μg/mL.

Each tube tested contains 80 μl of protein sample and 920 μL of Bradfordreagent (Sigma Aldrich product number B6916). After the Bradford reagentis added to each tube, the tubes are gently shaken to create a vortex,then incubated for 5 min at room temperature. The samples aretransferred to 1.5 mL cuvettes and their absorbance is measured at 595nm for 1h.

The protein concentration was determined by comparison with the standardprotein solutions prepared.

The measured protein amounts are:

-   -   before diafiltration: 295.45+/−9.91 μg/mL    -   after diafiltration: 160.61+/−1.31 μg/mL.

The electrophoretic profile of the tobacco leaf extract according toexample 1 was determined after the diafiltration step. Amounts of 2, 5,10 and 15 μg of proteins were deposited on a NuPage gel.

The 4%-12% Nupage gel was prepared for 35 minutes then placed in 20 mlof Coomassie Blue for 1 h and rinsed with water overnight. The gel wasanalysed using Biorad's ChemiDoc™ XRS system with the Image Lab™software.

Three main bands are detected around 15 kDa and 30 kDa (FIG. 1). Twoother bands are also perceptible at about 40 kDa.

The results show that the amount of proteins of molecular mass greaterthan 100 kDa detected is small compared with the amount of proteins ofmolecular mass between 10 and 50 kDa, in this tobacco leaf extract.

Example 4: Influence of Diafiltration Volume on the Removal of Moleculesof Molecular Mass Less than 10 kDa in a Tobacco Leaf Extract Accordingto the Invention

The removal of molecules of molecular weight less than 10 kDa in thetobacco leaf extract according to example 1 was monitored during thediafiltration step according to the diafiltration volume used.Monitoring is performed by gas chromatography assay of an analyticaltracer (nicotine) of molecular mass less than 10 kDa.

The results are presented in the following table:

Percentage of the Samples according to Peak area of the analyticaltracer diafiltration volume analytical tracer remaining Beforediafiltration 54913  100% Diafiltration x3 volume 2630 4.79%Diafiltration x4 volume 809 1.47% Diafiltration x5 volume 247 0.045% Diafiltration x6 volume NQ NQ

The removal of molecules of molecular weight less than 10 kDa isincreasingly effective when the diafiltration volume increases and istotal when this diafiltration volume is 6 times the initial volume.

Example 5: Biological Activity of the Extract of Example 1 In Vitro onHuman Mononuclear Cells and In Vivo on Mice

Materials and Methods:

Products Used

PMA/Ionomycin (Sigma-ALdrich product number P8139/10634)

BSA (Sigma ALdrich product number A7020)

PBS (PAA Laboratories product number H15-002)

Tween 20 (Sigma ALdrich product number P1379)

Goat anti-mouse IgG (g chain specific)-Alkaline Phosphatase (SouthernBiotech product number 1030-04)

Biotin-anti mouse IgE (Biolegend product number BLE406904)

Streptavidin—Alkaline Phosphatase (Southern Biotech product number7100-04)

Cell Preparation and Stimulation Mode

Peripheral blood mononuclear cells from different healthy donors wereisolated by centrifugation on a density gradient (separation medium: LMS1077 PAA Laboratories). The mononuclear cells obtained were stimulatedwith the extract according to example 1 at different dilutions for 24hor 48h. The supernatants were removed and stored at −80° C. after 24h or48h of stimulation for cytokine profile analysis using the Luminextechnique. After 24h of stimulation the cells were also taken forphenotypic analysis by flow cytometry.

Cytometric Phenotypic Analysis

The cells were first stained with a viability marker (Dye eFLuor 450(65-0863-14 eBioscience) and then with antibodies directly coupled tofluorochromes for the detection of different membrane markers. T and Blymphocyte populations were identified with markers CD3 and CD19. NKcells were detected either with marker NKp46 or the combination ofmarkers CD56 and CD16 or via CD56 expression in the absence of CD3. Fromthese cell populations, “gates” were set to analyse different activationmarkers (CD69, CD25, HLA-DR).

The List of antibodies used for the phenotypic cytometric analysis isgiven in the following table:

Clone Product number and supplier Anti-CD45-Krome Orange J.33 A96416;Beckman Coulter Anti-CD56 APC B159 555518; Becton Dickinson Anti-CD25PC7M-A251 557741; Becton Dickinson Anti-CD25 PC5 B1.49.9 IM2646; BeckmanCoulter Anti-CD69 FITC FN50 557049; Becton Dickinson Anti-CD69 PE L78341652; Becton Dickinson Anti-CD3 ECD UCHT1 A07748; Beckman CoulterAnti-KpP46 APC 9E2/NKp46 558051; Becton Dickinson Anti-CD19 FITC HIB1911-0199-42; eBioscience Anti-HLA-DR PE G46-6 347401; Becton Dickinson

Isotype controls were used in each experiment and a compensation matrixwas created for this multiparametric phenotypic analysis.

The stained samples were analysed on a Navios cytometer (Beckman Coulter10-colour) with a minimum acquisition of 100 000 events per tube. Theacquired data were then analysed on the Kaluza software.

Analysis of the Cytokine Secretion Profile by Luminex and ELISA

This technology, based on ELISA and cytometry principles, allows severalanalytes to be assayed simultaneously.

Unique colour-coded microbeads are coupled to capture antibodiesspecific for each analyte. After incubation with the samples to beassayed, detection antibodies coupled to a fluorochrome will allowanalysis by a dual-laser optical system (Bio-plex 200 system Bio-rad).The first Laser identifies the microbead and the second quantifies thedetection antibodies coupled to the fluorochrome. 17 analytes can beassayed with the Bio-PLex Pro Human Cytokine Grp1 panel 17 plex kit(Bio-rad) on samples taken during the various stimulation experimentsperformed on the cells. The cells stimulated by the culture mediumwithout the tobacco Leaf extract according to example 1 make it possibleto obtain the basal Level for each cytokine.

IL-8 ELISA is based on a conventional immunoenzymatic sandwichtechnique. The commercial kits used were purchased from Eurobio-Diaclone(Besançon).

Immunization of Mice with the Tobacco Leaf Extract According to Example1

Seven-week-old female C57BL/6 and Balb/c mice were purchased fromCharles River (L'Arbresles, France). The mice were immunized on days 0and 21 intraperitoneally with 200 μL of the tobacco leaf extractaccording to example 1 lyophilized and reconstituted in 5 mL of salinesolution. Blood was collected from all mice before and afterimmunization. After centrifugation, the serum was frozen at −20° C.

IgG and IgE ELISA

For the ELISA experiment, one vial of the product was reconstituted with2.5 mL of carbonate-bicarbonate buffer (0.1M, pH 9). Nunc Maxisorb 96Wplates were incubated with 100 μL of the tobacco leaf extractLyophilized according to example 1 then reconstituted and incubated for18h. After washing the plates with PBS-0.05% Tween buffer, the platesare saturated with 200 μL of PBS-1% BSA buffer for one hour at roomtemperature. After washing the plates with PBS-0.05% Tween buffer, 100μL of mouse serum, diluted to 1/20 in PBS-1% BSA, is incubated for 2hours at room temperature.

Elisa IgG:

After washing with PBS-Tween 20 buffer, 100 μL of a goat anti-mouse IgGsecondary antibody coupled to alkaline phosphatase (SouthernBiotechnology product number 1030-04) and diluted in PBS-1% BSA isincubated for 1 hour at temperature. After washes with PBS-0.05% Tween,100 μL of p-Nitrophenyl phosphate substrate (Sigma pNPP) of alkalinephosphatase is added and left 15 min at room temperature in the dark.The reaction is then stopped by adding 50 μL of 3M NaOH and the opticaldensities (OD) are read at 405 nm.

Elisa IgE

After washing with PBS-Tween 20 buffer, 100 μL of a rat anti-mouse IgEsecondary antibody coupled to biotin (Biolegend product numberBLE406904) and diluted in PBS-1% BSA is incubated for 1 hour at roomtemperature. After washes with PBS-0.05% Tween, 100 μL of streptavidincoupled to alkaline phosphatase (Southern Biotechnology product number7100-04) is added and incubated for 1 hour at room temperature (dilution112000^(th) in PBS-1% BSA according to the supplier's recommendations).After further washes with PBS-0.05% Tween, 100 μL of pNPP substrate ofalkaline phosphatase is added and left 15 min at room temperature in thedark. The reaction is then stopped by adding 50 μL of 3M NaOH and theoptical densities (OD) are read at 405 nm.

Results:

The Tobacco Leaf Extract According to Example 1 Causes a Specific IgGHumoral Response in Mice.

After 2 immunizations of the mice with the tobacco leaf extractLyophilized according to example 1 and reconstituted in 5 mL of salinesolution, IgG induction was observed against components of the product.Thus, 4/5 C57BL/6 mice and 4/5 Balb/c mice showed an increase in theirIgG against the tobacco leaf extract on day 28, i.e. 7 days after the2^(nd) vaccination (FIG. 2). The only mouse in each group which did notrespond had basal IgG antibody levels. Antibody titres appear lower inthe Balb/c mice than in the C57BL/6 mice (FIG. 2).

However, no IgE humoral response after 2 vaccinations of the mice withthe tobacco leaf extract was detected. Optical densities (OD) were lessthan 0.05 in all assays performed on the different sera.

The Tobacco Leaf Extract Preferentially Activates Peripheral BloodNatural Killer Cells

After isolation of mononuclear cells from peripheral blood by densitygradient (Ficoll), different cell populations (T and B lymphocytes, NKcells, etc.) can be characterized (FIG. 3A-3I). Thus, NK cells wereidentified by markers CD56 and CD16 in the CD3 negative population or bymarker NKp46. After incubation of the mononuclear cells with the tobaccoleaf extract according to example 1, an increase in expression ofactivation markers CD69, CD25 and HLA-DR was observed on the NK cells,identified both by markers CD16+CD56+ (CD3−) (FIG. 4A) and by markerNKp46 (FIG. 4B). The results are more distinct with the tobacco leafextract diluted to ½ and for CD69 expression. Thus, more than 60% of NKcells express marker CD69 24 hours after contact with the tobacco leafextract (FIG. 4A-B). At rest, less than 5% of blood NK cells expressCD69.

The effect of the tobacco leaf extract on activation of peripheral bloodlymphocytes is much weaker. Thus, at rest 2% of T lymphocytes expressCD69 and this expression increases to 6% after 24 hours of culture withthe tobacco leaf extract (FIG. 5A). CD25 expression is not increased onT lymphocytes sensitized with the tobacco leaf extract (FIG. 5A).Similarly, on peripheral blood B lymphocytes, the tobacco leaf extracthas no significant activity, as less than 3% of B lymphocytes expressCD69 after 24 hours of co-culture with this extract (FIG. 5B).

Profile of Cytokines Induced by the Tobacco Leaf Extract According toExample 1 on Human Blood Mononuclear Cells

Peripheral blood mononuclear cells were brought into contact for 24hours with the tobacco leaf extract lyophilized according to example 1and reconstituted with 5 mL of PBS at different dilutions (1/2 or 1/20)and assays of cytokines, chemokines and growth factors were performed byLuminex in the supernatant.

FIG. 6 shows a strong induction of pro-inflammatory cytokines (IL-1β,IL-6, TNFα, IL-17) by the tobacco leaf extract. For example, the basallevels of these cytokines when the mononuclear cells are not placed inthe presence of the tobacco leaf extract are less than 10 pg/mL (FIG.6A).

Concentrations of cytokines IL-1B, IL-6 and TNFα are measured at over 1000 pg/mL after incubation with the tobacco leaf extract diluted to ½(FIG. 6A). This clear induction of pro-inflammatory cytokines is alsoobserved when the tobacco leaf extract is diluted to 1/20 (FIG. 6A). Theresults are fairly homogeneous between the different donors tested.Among the TH1 cytokines (IL-2, IFNγ, IL-12) promoting cell-mediatedimmunity, only IFNγ is significantly stimulated by the tobacco leafextract diluted to 1/2 and to 1/20 (FIG. 6B). The tobacco leaf extractinduces very low concentrations of TH2 cytokines (IL-4, IL-5, IL-13) ofthe order of a few pg/mL (FIG. 7A). In contrast, the tobacco leafextract stimulates IL-10 production by mononuclear cells with levels of750 pg/mL found in the supernatant of cells brought into contact withthe tobacco leaf extract diluted to 1/2 (FIG. 7A). IL-10 was originallyconsidered a TH2 cytokine but it can also be produced by regulatory Tlymphocytes called Tr1.

We have shown that the tobacco leaf extract can also induce theproduction of hematopoietic growth factors such as G-CSF which promotesthe expansion and recruitment of neutrophils and chemokines such asMCP-1 known to exert a chemotactic effect on macrophages (FIG. 7B).However, the tobacco leaf extract does not appear to significantlystimulate other growth factors such as IL-7 or GM-CSF (FIG. 7B). Thesame cytokine/chemokine profile was found when the assays were performedon supernatants collected 48 hours after culture with the tobacco leafextract.

CONCLUSION

The tobacco leaf extract according to example 1 is capable of inducing aspecific IgG humoral response directed against proteins present in theproduct in two mouse lines of different genetic background.

No IgE humoral response was observed after two administrations of thesubstance in these same mice.

In vitro, the tobacco leaf extract according to example 1 preferentiallyinduces the activation of NK cells among peripheral blood mononuclearcells. This activation is reflected by the increased expression of CD69,CD25 and HLA-DR molecules.

The tobacco leaf extract according to example 1 induces in vitropro-inflammatory cytokines such as IL-1β, IL6, IL-17, IL-8 and TNFα.These cytokines are present in high concentrations in the culturesupernatant collected 24 hours after stimulation.

The tobacco leaf extract according to example 1 also induces IFNγ,IL-10, G-CSF and MCP-1. However, this extract does not significantlyinduce the production of TH2 cytokines (IL-4, IL-5, IL-13).

Example 6: Toxicological Study of a Tobacco Leaf Extract According toExample 1

Materials and Methods:

All studies below were performed with the tobacco leaf extract accordingto example 1. The route of administration is subcutaneous in all cases.The toxicology studies were conducted on rodents (Han Wistar rats). Thetoxicological studies were carried out according to the OECD principlesof good aboratory practice (Mutual Acceptance of Data (MAD) in theassessment of data, 26 Nov. 1997 (C(97) 186 Final) and according to theGood Laboratory Practices (GLP) published by the French Ministry ofEmployment and Solidarity (No 2000/5 bis, Order of 14 Mar. 2000, JO23/03/2000).

Study of Subcutaneous Toxicity at 14 Days

The objective of this study was to determine the toxicity of the tobaccoleaf extract according to example 1 in rats following subcutaneousadministration (once a week for 2 weeks).

The study was conducted as follows:

Dose con- centration Dose expressed expressed Group as protein Dosevolume as protein Number of animals No (μg/kg/adm) (mL/kg/adm) (μg/mL)males females 1 0 2 0 6 6 2 11.2 0.4 28 6 6 3 56 2 28 6 6

The results of the study showed that:

-   -   no mortality is observed at any dose;    -   no treatment-related clinical signs are observed and        subcutaneous administration is well tolerated locally (clinical        signs and local tolerance were observed before and after        injection, and once daily when no treatment is administered);    -   the effects on body weight and food consumption (compared to        controls) are not harmful;    -   there are biologically significant differences in white blood        cell counts between treated and untreated rodents but these        differences have no toxic effect;    -   variations were observed between the treated and untreated        groups in terms of protein and cholesterol concentrations, but        these variations remained within or close to those of the        control group. These effects are not harmful;    -   no change was observed in organ weights between the treated        group and the control group.

Subcutaneous administration to Wistar rats of the tobacco leaf extractaccording to example 1 once weekly for 2 weeks at doses of 11.2 and 56μg protein/kg/adm was therefore well tolerated.

Four-Week Study of Subcutaneous Toxicity

The objective was to determine the toxicity of the tobacco leaf extractaccording to example 1 following subcutaneous administration once weeklyto Wistar rats and to determine the regression of any sign of toxicityover a period of 4 weeks without treatment.

Particular attention was paid to potential immunological phenomena.

The study was conducted as follows:

Number of animals After Dose Dose After observation Dose⁽¹⁾ volumeconcentration treatment⁽²⁾ period⁽³⁾ Group/treatment (μg/kg/adm)(mL/kg/adm) (μg/mL) males females males females 1. Control 0.0 4.0 0.010 10 5 5 (vehicle: 0.9% NaCl) 2. low dose 16.8 0.6 28.0 10 10 / / 3.intermediate 33.6 1.2 28.0 10 10 / / dose 4. high dose 112.0 4.0 28.0 1010 5 5 ⁽¹⁾Expressed as proteins ⁽²⁾Rats killed 2 days after the lastadministration (day 23) ⁽³⁾Rats killed 4 weeks after the lastadministration (day 49)

During the treatment period, the rats were observed before and at least3 times after administration. During the observation period, the ratswere observed once daily.

A complete clinical examination was performed once a week. Localtolerance was noted before and after each injection, once daily duringthe first week and then twice weekly until the end of the observationperiod.

Body temperature was measured the day after each administration (days 1,8, 15 and 22).

Ophthalmologic examinations were performed before the test, as well asone day after the first and last day of administration (days 1 and 22).

Individual body weights and food consumption were measured twice a week.

Haematological, coagulation, serum clinical chemistry parameter andlymphocyte analyses were performed on days 23 and 49/48 (male/female,respectively). Urine tests were performed on day 23.

All animals were killed 2 days after the last administration or after a4-week observation period and autopsied. Certain organs were weighed andused for histopathological examinations.

The results of the study showed that:

-   -   no mortality is observed at any dose;    -   subcutaneous administration at doses expressed as protein of        16.8, 33.6 and 112.0 μg/kg/adm was well tolerated locally;    -   no treatment-related clinical signs are observed;    -   body temperature is not affected;    -   no treatment-related ophthalmologic effect was observed;    -   body weight and food consumption are not affected;    -   no significant differences in terms of haematology, coagulation        and urine were observed;    -   creatine kinase enzyme activity decreased in female groups 3 and        4 (intermediate and high doses; 33.6 and 112 μg/kg/adm,        respectively) at the end of the treatment period. These        variations were not considered harmful insofar as they are not        associated with histopathological features;    -   a slight decrease in the number of circulating NK cells in        treated females and in males treated with intermediate and high        doses (groups 3 and 4);    -   at the end of the observation period, the mean number of        circulating NK cells in males treated with a high dose (group 4)        was lower;    -   no change in organ weight was observed;    -   a dark spot was observed at the injection site, which        corresponds to subcutaneous bleeding during histopathological        evaluation. This observation is considered to be unrelated to        the administration procedure.

At the end of treatment, minimal or slight inflammatory skin changeswere observed at the injection site of groups 3 and 4, while almost noinflammatory change was observed for groups 1 and 2.

At the end of the observation period, the inflammatory changespreviously observed for group 4 showed an almost total recovery.

The 4 subcutaneous injections of the tobacco leaf extract according toexample 1 up to a dose of 112 μg/kg/adm were well tolerated and onlyminimal and reversible inflammatory changes occurred in treated ratscompared with untreated rats.

In conclusion, under the experimental conditions defined above,subcutaneous administration in Wistar rats of the tobacco leaf extractaccording to example 1 once weekly for 4 weeks at doses of 16.8, 33.6and 112 μg/kg/adm was well tolerated and not associated with any adverseeffects.

Example 7: Clinical Study Following Administration of a Tobacco LeafExtract According to Example 1

A pharmaceutical composition comprising the tobacco leaf extractaccording to example 1, containing 100 μg or 200 μg of proteins andmannitol, was administered by subcutaneous injection a first time on day0 and a second time on day 29 (1 mL in each arm each time) to 24smokers. Inclusions were made according to the principle of cohortexpansion, 6 initial patients receiving 100 μg being included first,followed by 6 initial patients receiving 200 μg, before the inclusion of6 additional patients receiving 100 μg. The population of subjectstreated consisted of 24 people, 14 men and 10 women, between 30 and 65years of age with a median age of 44.5 years.

It should be noted that none of the doses tested generated a toxicity oflevel 3-4 according to the toxicity grades of the Common TerminologyCriteria for Adverse Events (CTCAE) v4.0 of the National CancerInstitute.

The initial characteristics of the subjects in terms of tobaccoconsumption are presented in the table below.

N % Median Range Consumer status Smoker 24 100 Consumption Manufacturedcigarettes, No/day 18 75 20 (15; 30) Rolled cigarettes, No/day 3 12.5 20(15; 30) Cigarillos, No/day 3 12.5 20 (15; 20) Fagerström dependencelevel 5 11 46 6 5 21 7 2 8 8 3 12.5 9 3 12.5 Exhaled CO Measurement(ppm) 24 24.5  (9; 45) Would you like to quit smoking? No Yes 24 100

FIGS. 8 and 9 show the evolution of tobacco consumption.

The formula for calculating the decrease in number of cigarettes is asfollows: (No of cigarettes visit n−No of cigarettes at inclusion)/No ofcigarettes at inclusion.

A point made at 4 weeks showed that 16 smokers (67% of the smokers) hadquit smoking or reduced their daily cigarette consumption by at least50% and that the daily cigarette consumption of the 24 smokers haddecreased by 60%.

A point made at 12 weeks showed that 15 smokers (63% of the smokers) hadquit smoking or reduced their daily cigarette consumption by at least50% and that the daily cigarette consumption of the 24 smokers haddecreased by 60%.

It is interesting to note that the last 12 smokers (12 out of 24)benefited from a longer delay (sometimes a month or more) between theirfirst contact with the clinical study centre and the injection of atobacco extract according to example 1, whereas the first 12 smokers hada shorter delay (often only a few days). For these 12 smokers whobenefited from a longer delay, the percentage of smokers who reducedtheir consumption by at least 50% or stopped smoking at 3 and 4 weeks is83% (10 smokers out of 12) whereas it is 50% (6 smokers out of 12) forthe 12 smokers who benefited from a shorter delay. This superiorefficacy is explained by the better preparation of those smokers who hadmore time to reinforce their motivation before receiving an injection ofa tobacco extract according to example 1. Of the last 12 smokers, 5 wereabstinent continuously from one week after the end of treatment to week12 (end of follow-up), and 8 were continuously abstinent over the last 7days at week 12.

The treated smokers describe a reduction in their attraction tocigarettes and their urge to smoke as well as the institution of acertain indifference to cigarettes. They notice a reduction in thepalatability of cigarettes. Some speak of a change in the taste ofcigarettes. Their motivation to quit is reinforced.

Before administration of the pharmaceutical composition, IgG antibodiesdirected against the pharmaceutical composition were detected in allpatients. Before treatment, the mean concentrations of IgG directedagainst the pharmaceutical composition were 6.07 microg/ml (standarddeviation: 2.98 microg/ml).

After administration of the pharmaceutical composition, a significantincrease in concentrations of anti-pharmaceutical composition IgG wasobserved. Thus, the IgG concentrations measured 29 days afteradministration of the pharmaceutical composition were 7.19 microg/ml(standard deviation: 4.04 microg/ml) and these concentrations increasedto 7.5 microg/ml (standard deviation 4.28 microg/ml) when measured 8weeks after the first administration of the pharmaceutical composition.Variations in IgG concentrations were significant (t-test significancethreshold p<0.05) between day 29 and day 0 (p=0.02) and between themeasurements of week 8 and day 0 (p=0.006). A significant difference wasalso observed between the concentrations of anti-pharmaceuticalcomposition IgG at week 8 and at day 29 (p=0.029).

Statistical analysis crossing the concentrations of IgG directed againstthe pharmaceutical composition and smoking cessation showed correlations(Wilcoxon test, significance threshold p<0.05) between higher levels ofIgG directed against the pharmaceutical composition and smokingcessation.

Patients who reduced their consumption by at east 50% one week after thefirst administration of the pharmaceutical composition had higher levelsof IgG directed against the pharmaceutical composition at day 29(p=0.034) and at week 8 (p=0.044).

Patients who were abstinent for 7 consecutive days (prevalentabstinence) at day 36 had higher levels of IgG directed against thepharmaceutical composition at day 29 (p<0.001) and at week 8 (p<0.001).

Patients who were abstinent for 7 consecutive days (prevalentabstinence) at week 12 had higher levels of IgG directed against thepharmaceutical composition at day 29 (p=0.013) and at week 8 (p=0.016).

Patients who were continuously abstinent until week 12 had higher levelsof IgG directed against the pharmaceutical composition at day 29(p=0.007) and at week 8 (p=0.009).

IgE antibodies were not detected following administration of thepharmaceutical composition.

The invention claimed is:
 1. A tobacco leaf extract containing at least5% by weight, based on the total weight of the dry extract, of proteinsof molecular mass greater than 10 kDa, and essentially free of moleculesof molecular mass less than 10 kDa and of RuBisCO proteins, wherein thecontent of proteins whose molecular mass is greater than 500 kDa is lessthan 15% by weight based on the total protein weight of the extract. 2.The tobacco leaf extract according to claim 1, wherein the proteins areselected from the group consisting of the following protein families:lignin-forming anionic peroxidase, glucan endo-1,3-beta-glucosidase,endochitinase, pathogenesis-related protein, osmotin, proteinaseinhibitors, and mixtures thereof.
 3. The tobacco leaf extract accordingto claim 1, wherein the content of proteins whose molecular mass isgreater than 100 kDa is less than 15% by weight based on the totalprotein weight of the extract.
 4. The tobacco leaf extract according toclaim 1, comprising at least one protein belonging to the family ofglucan endo-1,3-beta-glucosidases selected from the group consisting ofbeta-1,3-endoglucanase acidic isoform PR-Q′ (PR36401 according to theUniProt database), beta-1,3-endoglucanase basic vacuolar isoform GLB(P27666 according to the UniProt database), and mixtures thereof.
 5. Thetobacco leaf extract according to claim 1, comprising at least oneprotein belonging to the family of endochitinases selected from thegroup consisting of acidic endochitinase P (P17513 according to theUniProt database), acidic endochitinase Q (P17514 according to theUniProt database), endochitinase B (P24091 according to the UniProtdatabase), and mixtures thereof.
 6. The tobacco leaf extract accordingto claim 1, comprising at least osmotin (P14170 according to the UniProtdatabase).
 7. The tobacco leaf extract according to claim 1, comprisingat least one lignin-forming anionic peroxidase (P11965 according to theUniProt database).
 8. The tobacco leaf extract according to claim 1,comprising at least one pathogenesis-related protein selected from thegroup consisting of pathogenesis-related protein R (P13046 according tothe UniProt database), pathogenesis-related protein PR-4A (PR29062according to the UniProt database), pathogenesis-related protein PR-4B(PR29063 according to the UniProt database), and mixtures thereof. 9.The tobacco leaf extract according to claim 1, comprising at least oneprotein belonging to the family of proteinase inhibitors selected fromthe group consisting of proteinase inhibitor I-B (Q03199 according tothe UniProt database), proteinase inhibitor I-A (Q03198 according to theUniProt database), and mixtures thereof.
 10. The tobacco leaf extractaccording to claim 1 wherein the protein content in the dry extract isat least 10% by weight based on the total weight of the dry extract. 11.A pharmaceutical composition comprising as active principle a tobaccoleaf extract according to claim 1 as well as a pharmaceuticallyacceptable excipient.
 12. The pharmaceutical composition according toclaim 11, wherein the proteins present in said tobacco leaf extract arepresent in an amount ranging from 1 to 1 000 μg/mL.
 13. Thepharmaceutical composition according to claim 11, characterized in thatit further contains proteins extracted from cannabis.
 14. Thepharmaceutical composition according to claim 11, which is provided in aform suitable for administration by subcutaneous injection, in a formsuitable for administration by means of an adhesive transdermaltherapeutic system such as a patch, or in a form suitable foradministration by spraying or by vaporization.
 15. A tobacco leafextract containing at least 5% by weight, based on the total weight ofthe dry extract, of proteins of molecular mass greater than 10 kDa, andessentially free of molecules of molecular mass less than 10 kDa and ofRuBisCO proteins; wherein the content of proteins whose molecular massis greater than 500 kDa is less than 15% by weight based on the totalprotein weight of the extract; and wherein the tobacco leaf extractpromotes smoking cessation after administration to a subject in needthereof.